ABSTRACT The myokine irisin is a peptide that is proteolyzed from the muscle-bound protein Fndc5 (fibronectin type III domain-containing protein 5) during exercise and enters circulation and induces a thermogenic program in beige adipose tissue1,2. Several studies have suggested a key role for irisin in mediating the effect of exercise on bone, demonstrating that intermittent low dose irisin (0.1 mg/kg once per week) stimulates cortical bone formation and prevents unloading-induced bone loss in vivo, and enhances osteogenesis in vitro3-5. Utilizing the power of mouse genetics, our group has endeavored to further elucidate the role of irisin in skeletal remodeling, demonstrating that forced expression of Fndc5 in muscle markedly reduces bone formation and mass, decreases osteoblast number while increasing osteoclasts, and increasing NF-?B and SOST expression while suppressing serum levels of bone formation markers. Similarly, genetic deletion of Fndc5 led to high vertebral bone volume and complete protection from ovariectomy (OVX)-induced bone loss in female mice, marked by maintenance of osteocyte lacunae density and size, blocked bone resorption, and no increase in RANKL expression despite prolonged estrogen deprivation. Short term irisin infusions in wild type mice resulted in higher serum levels of sclerostin and greater SOST mRNA expression. Irisin treatment of MLOY-4 osteocytes in vitro demonstrated a direct effect on this cell type, inducing gene and protein level expression of sclerostin in a dose dependent manner and preventing hydrogen peroxide-induced apoptosis. Importantly, we identified through biochemical and biophysical means that the ?V?5 integrin is the principal, although possibly not the only, receptor for irisin in osteocytes6. We now have evidence that in addition to its effect on the osteocyte, irisin acts directly on the osteoclast to stimulate differentiation, and this effect is reversed by blocking both ?V?5 and ?V?3 with antibody antagonists. At face value these data would seem to run counter to the prevailing hypothesis that myokines are purely anabolic for bone. However, in a physiologic context irisin might also indirectly stimulate osteoblasts via release of clastokines or matrix-bound growth factors, or it may have a unique role as a counter regulatory hormone to maintain calcium homeostasis by increasing resorption. With the work proposed herein, we will address two specific aims to test the central hypothesis that irisin acts as a key regulating factor in the influence of exercise on bone remodeling; both by direct action on the osteoclast through integrin ?V?5, and by modulation of osteoclast paracrine signaling with osteocytes and osteoblasts. Because this myokine may play a key role in linking physical activity and bone remodeling, this work will focus both on traditional static in vitro culture models as well as address the effect of mechanical loading such as fluid shear on the osteoclast, osteoblast, and osteocyte with specific regard to the influence of irisin on cell mechanosensitivity.